Drug preparation

4-Hydroxytamoxifen preparation

1. Weigh 50 mg 4-Hydroxytamoxifen (Sigma: #H6278) into a 15 mL falcon tube, then add 2.5 mL ethanol into the tube. Seal the cap and vortex the suspension for ~1 min Alternatively, add 2.5 mL ethanol into the Sigma bottle if it can accommodate that volume.
2. Place the tube on a horizontal shaker, make sure it is protected from light. Shake the tube at room temperature until 4-Hydroxytamoxifen dissolves, which usually takes ~15 minutes.
3. Aliquot the 4-Hydroxytamoxifen stock solution (20 mg/mL) into 1.5 mL Eppendorf tubes and store them in -20°C degree (it is common to observe 4-Hydroxytamoxifen precipitation in -20°C).
4. On the day of 4-Hydroxytamoxifen administration, freshly prepare a 4-Hydroxytamoxifen working solution. Take an aliquot of 4-Hydroxytamoxifen stock (20mg/mL)from -20°C, wrap the stock solution with aluminum foil, warm it up to room temperature, and shake on horizontal shaker until precipitation re-dissolves (it usually takes 30 min).
5. Once 4-Hydroxytamoxifen completely dissolves, add the desired amount of corn oil (Sigma: #C8267) into the tube to obtain a final concentration of 10 mg/mL (working solution). For example, if the stock aliquot is 200 µl, then add double the volume of corn oil i.e. 400 µl.
6. the corn oil and 4-Hydroxytamoxifen solution was well mixed.
7. Place the oil:ethanol mixture into a vacuum centrifuge (SpeedVac), and centrifuge the tube with vacuum on to evaporate the ethanol. The duration of centrifuge depends on the volume of mixture and power of vacuum. It usually takes 10-20 min for the ethanol to fully evaporate.
8. Take the tube out of centrifuge, the solution should be clear with no apparent delamination or precipitation.
9. Weigh the animal before drug administration and load desired volume of 4- Hydroxytamoxifen solution into a BD insulin syringe.
10. Administer the 4-Hydroxytamoxifen solution through intraperitoneal injection so that the final dosage of 4-Hydroxytamoxifen is 50 mg/kg.

Note: please always keep the 4-Hydroxytamoxifen protected from light; The 4-Hydroxytamoxifen working solution should be freshly prepared.
 

Clozapine N-oxide preparation

1. Calculate the amount of Clozapine N-oxide (Tocris, #4936) and weigh it into a Eppendorf tube.
2. Dissolve in DMSO to obtain 5 mg/mL Clozapine N-oxide solution, it should be clear yellow solution. Protect the solution from light.
3. Weigh the animal before drug administration and load desired volume of Clozapine N- oxide solution into a BD insulin syringe.
4. Inject the Clozapine N-oxide solution intraperitoneally at final dosage of 5 mg/kg body weight 1 hr prior to behavioral manipulation.

Note: please always keep the Clozapine N-oxide protected from light; The Clozapine N-oxide working solution should be freshly prepared.